1. Field of the Invention
The present invention relates to a process for preparing neotrehalose and its uses, more particularly, it relates to a preparation of neotrehalose comprising allowing .beta.-galactosidase (EC 3.2.1.23) to act on an aqueous solution containing lactoneotrehalose and recovering the resultant neotrehalose, as well as to orally-administrable products containing said neotrehalose.
2. Description of the Prior Art
Neotrehalose, which is a disaccharide shown by the formula O-.alpha.-D-glucopyranosyl .beta.-D-glucopyranoside or O-.beta.-D-glucopyranosyl .alpha.-D-glucopyranoside, is a well known compound. Neotrehalose has a sweetness, non-reducibility, satisfiable stability, and high water-solubility, and these merits render neotrehalose very useful in the preparations of orally-administrable products, for example, foods, beverages, cigarettes and tobaccos.
Although methods to prepare neotrehalose, for example, those using chemical syntheses as reported in W. N. Haworth et al., Journal of the Chemical Society, pp.2847-2850 (1931), and in V. E. S. Sharp et al., ibid, pp.285-288 (1951), have been known, the yield of neotrehalose is extremely low and there exist many problems which should be overcome in view of the safeness, prior to actual use.
The preparations of neotrehalose from natural substances with a greater safety or those by using biochemical syntheses have been proposed. For example, K. Matsuda reported in Journal of the Agricultural Chemical Society of Japan, Vol. 30, No. 2, pp. 119-123 (1959) that a small amount of neotrehalose was present in a koji extract together with isomaltose and kojibiose: In this report, neotrehalose was prepared by a method comprising subjecting a koji extract to a column chromatography using activated charcoal to remove non-fermenting saccharides in the extract, recovering fractions containing disaccharides, treating the disaccharides with phenylhydrazine, separating the resultant sediment to obtain a filtrate, acetylating the saccharides in the filtrate to form crystalline octaacetates of the disaccharides, and deacetylating the crystalline octaacetates. S. Kobayashi et al. disclosed in Japanese Patent Laid-Open No. 216,492/88 a method containing a step of allowing a cyclodextrin-forming enzyme to act on amylaceous substance to form neotrehalose together with centose: In this report, high-performance liquid chromatography (HPLC) revealed that the reaction solution contained 18.6% neotrehalose and 20.0% centose, and paper chromatography analysis revealed that the resultant neotrehalose-rich fraction contained about 20% of nigerose, kojibiose and isomaltose. S. Kobayahshi et al. also proposed a method to prepare neotrehalose from the reaction solution comprising heating the reaction solution to inactivate the remaining cyclodextrin-forming enzyme, adding to the resultant solution glucoamylase and yeast at the same time to digest and remove the forming glucose, removing the remaining yeast, autoclaving the resultant after the addition of sodium hydroxide, neutralizing the resultant product, and recovering the resultant neotrehalose with column chromatography and/or the sedimentation method using solvents. None of the methods has as yet been industrially carried out because of the following reasons: (i) The methods only produce neotrehalose preparations with a relatively low-content of neotrehalose; and (ii) The purification steps required after the methods are extremely complicated, and the yield of neotrehalose attained thereby is relatively low.